A number of water-soluble (interfacial) proteins involved in cell signaling do so by binding to phospholipid membranes to produce products at the membrane surface. Such proteins control various cellular responses, such as the release of arachidonic acid for biosynthesis of eicosanoids, second messenger liberation for calcium signaling, and cell-surface triggered blood coagulation. The number of high-resolution X-ray structures of interfacial proteins is increasing, and is providing an understanding of the structural motifs used for membrane binding. However, such studies do not give a molecular picture of how membrane binding motifs interact with phospholipid bilayers nor the nature or extent of structural transitions that accompany membrane binding. Our proposed studies using advanced electron paramagnetic resonance (EPR) spectroscopy, novel spin relaxant methods that we have developed, and site-selective spin-labeling of interfacial proteins will build on and complement the X-ray crystallographic work by providing not only the orientation of the interfacial proteins with respect to the membrane but also the depth of penetration of the interfacial binding motifs into the membrane bilayer. In addition, the proposed studies will define structural transitions that are hypothesized to accompany membrane binding and they will set the stage for future studies aimed at defining the molecular mechanisms of enzyme catalysis. The current EPR studies focus on three important proteins that bind to the membrane for cell signaling: human cytosolic phospholipase A2, the d1 isoform of human phosphatidylinositol-specific phospholipase C, and the membrane-binding portion of factor VIII, which is part of the "intrinsic ten-ase complex". Determining the protein-membrane orientation and depth of protein penetration into the membrane will provide a molecular understanding of the roles of specific amino acid residues to promote interfacial binding to membranes of specific phospholipid composition. The current studies on three structurally well-characterized enzymes will provide important insights related to a long-term goal for this project which is to understand the energetics that drive membrane insertion of interfacial enzymes. This information is important in understanding the action of these proteins in cells or on cell surfaces because the function of these enzymes is controlled in part by their targeting to specific cell membranes.